Fat mass, adipocyte metabolic responsiveness, and capacity of preadipocytes to differentiate into fat cells decline with aging. Differentiation-dependent gene expression is blunted in preadipocytes isolated from old compared to younger individuals, even after several generations ex vivo. Our long-term objective is to elucidate mechanisms responsible for decreased adipogenesis with aging. In the first 3 years of this grant, we found lower CCAAT enhancer binding protein alpha (C/EBPalpha) expression in differentiating preadipocytes cultured from old than younger rats. Overexpression of C/EBPalpha in preadipocytes from old rats restored differentiation. Expression of the other key adipogenic regulator, peroxisome proliferator-activated receptor gamma (PPARgamma), also declined with aging and overexpression in old preadipocytes restored differentiation. Expression of the C/EBP family members C/EBP beta-LIP and C/EBPzeta (CHOP), both dominant negative inhibitors of adipogenesis, increased with aging, as did preadipocyte TNFalpha and IL6 release. These cytokines inhibit PPARgamma and C/EBPalpha activities. Based on these findings, our hypothesis is that age-related increases in cytokine release and activity of anti-adipogenic C/EBP family members interact during initiation of differentiation to inhibit adipogenesis causing altered fatty acid handling. We will test this hypothesis in the following aims. Aim 1 is to test the hypothesis that preadipocyte cytokine release contributes to the decline in adipogenesis with aging. We will test effects of aging changes in cytokine release on adjacent cells. Using transfection and pharmacological methods in preadipocytes from rats and humans of different ages, we will determine: 1) effects of aging on preadipocyte cytokine release, 2) the impact of this on adipogenesis, and 3) responsible mechanisms. Aim 2 is to test the prediction that increased expression of LIP and CHOP during initiation of differentiation contributes to decreased adipogenesis. We found that CUGBP, a protein that causes LIP to be translated from C/EBPbeta mRNA, increases with aging and TNFalpha treatment. Using preadipocytes cultured from individuals of different ages, we will test the hypothesis that increased TNFalpha stimulates CUGBP expression, causing elevated LIP with aging, inhibiting adipogenesis. We will also test the hypothesis that activation of the cellular stress response (stress activated protein kinase (SAPK) 1 and 2 activities) ultimately mediates increased CUGBP, LIP, and CHOP expression with aging. Aim 3 is to test the hypothesis that impaired adipogenesis, resulting in decreased levels of adipocyte fatty acid binding protein (aP2), leads to altered fatty acid handling. We found that processes accounting for reduced adipogenesis with aging are associated with reduced aP2 expression with physiologically relevant effects on fatty acid handling. We will test this association by manipulating aP2, adipogenic regulator, and cytokine levels and determining effects on fatty acid transfer and lipolysis in preadipocytes from individuals of different ages. These studies will contribute to understanding about mechanisms of age-related changes in fat cell function and differentiation.